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Updated: May 10, 2026

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Published on: September 25, 2019

Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor.

Deborah Cipria¹, Tania Baccega^1,2, Miriana Rizzo¹

¹San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Nature Communications

|May 8, 2026

Summary

This summary is machine-generated.

This study introduces a novel CRISPR-Cas9 epigenome editor for safe, precise cell engineering. The polyfunctional system allows simultaneous gene editing and silencing without causing harmful chromosomal translocations.

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Area of Science:

CRISPR-Cas9 gene editing
Epigenome engineering
Cancer immunotherapy

Background:

Orthogonal editing enables precise cell engineering but risks chromosomal translocations due to multiple DNA breaks.
Existing methods for simultaneous gene insertion and epigenetic modification are limited.

Purpose of the Study:

To develop a safe and efficient polyfunctional CRISPR-Cas9-based epigenome editor.
To enable simultaneous transgene insertion and epigenetic silencing at distinct genomic loci in a single treatment.
To avoid inducing reciprocal chromosomal translocations during multiplexed genome and epigenome engineering.

Main Methods:

An optimized all-in-one epigenome editor utilizing catalytically active Cas9.
Selective disabling of Cas9 endonuclease activity at silenced loci using truncated guide RNAs (gRNAs).
Demonstration in primary human T cells for multi-locus editing and silencing.

Main Results:

Efficient multi-locus editing, including TCR replacement and CAR insertion into specific loci.
Targeted insertion of selectable markers or immunomodulatory receptors.
Durable, multiplexed epigenetic silencing of clinically relevant genes without chromosomal translocations.

Conclusions:

The polyfunctional editing platform provides a versatile and safe framework for orthogonal genome and epigenome engineering.
This approach broadens the scope of cell engineering for applications in cancer immunotherapy and beyond.
The developed system overcomes safety concerns associated with multiple DNA breaks in advanced gene editing strategies.